1 Title: Molecular evidence for zoonotic transmission of Giardia duodenalis among dairy farm 1
workers in West Bengal, India 2
3
Shahbaz Manzoor Khana, b, Chanchal Debnathb, Amiya Kumar Pramanikb, Lihua Xiaoc, Tomoyoshi 4
Nozakid and Sandipan Gangulya,*. 5
6 7
a Division of Parasitology, National Institute of Cholera and Enteric Diseases, P-33, C. I. T. Road, 8
Beliaghata, Scheme-XM, Kolkata-700 010, West Bengal, India.
9 10
b West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Belgachia, Kolkata-700 11
037, West Bengal, India.
12 13
c Centers for Disease Control and Prevention, Atlanta, USA.
14 15
d Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan 16
17
* Corresponding author: - Dr. Sandipan Ganguly. Tel.: +91 33 23633855; Fax: +91 33 23632398;
18
email address: [email protected] 19
20
Note: Nucleotide sequence data reported in this paper are available in the GenBank™ database 21
under the following accession numbers: GQ290390, GQ345009 and GQ345010.
22 23 24 Manuscript
2 Abstract
25
No study in the past has examined the genetic diversity and zoonotic potential of Giardia 26
duodenalis in dairy cattle in India. To assess the importance of these animals as a source of human 27
G. duodenalis infections and determine the epidemiology of bovine giardiasis in India, fecal 28
samples from 180 calves, heifers and adults and 51 dairy farm workers on two dairy farms in West 29
Bengal, India were genotyped by PCR-RFLP analysis of the β-giardin gene of G. duodenalis 30
followed by DNA sequencing of the PCR products. Phylogenetic analysis was carried out on the 31
DNA sequences obtained in the study and those available in GenBank. The overall prevalence of 32
G. duodenalis in cattle was 12.2% (22/180), the infection being more prevalent in younger calves 33
than in adult cattle. Zoonotic G. duodenalis Assemblage A1 was identified in both calves and 34
workers although the most prevalent genotype detected in cattle was a novel Assemblage E 35
subgenotype. These findings clearly suggest that there is a potential risk of zoonotic transmission of 36
G. duodenalis infections between cattle and humans on dairy farms in India.
37 38
Keywords: Giardia duodenalis, Cattle, dairy farm workers, zoonoses, India, Genotyping.
39 40 41
3 1. Introduction
42
Protozoan parasites of the genus Giardia have a worldwide distribution and are emerging as 43
one of the most frequent causes of diarrhoea in humans in both the developing and developed 44
world. Giardiasis contributes to diarrhoea and nutritional deficiencies in children less than 10 years 45
of age with the highest prevalence in developing countries (Islam, 1990). In addition to humans, 46
they infect a wide variety of domesticated and wild animals having emerged as important parasites 47
of dairy cattle because of their proven pathogenicity (Xiao et al., 1993; Ruest et al., 1997;
48
O’Handley et al., 1999), and the potential public health significance of zoonotic transmission 49
(Buret et al., 1990; Ey et al., 1997; Olson et al., 2004).Giardia duodenalis (syn G. intestinalis, G.
50
lamblia) has been implicated as an etiological agent in dairy and beef calf diarrhoea, worldwide 51
(O’Handley et al., 1999; Huetink et al., 2001; Olson et al., 2004). In fact, reduced rate of weight 52
gain, impaired feed efficiency and decreased carcass weight were associated with giardiasis in a 53
ruminant model of the disease (Olson et al., 1995).
54
G. duodenalis is the only species found in humans, although it is also found in other 55
mammals, including pets and livestock (Thompson et al., 2000). Substantial evidence suggests G.
56
duodenalis to be a species complex comprised of morphologically indistinguishable isolates which 57
can genetically be differentiated into several major assemblages: Assemblages A and B mainly 58
infect humans but are also found in a wide range of other mammals; C and D have been found to 59
infect dogs; E has been isolated from livestock (cattle, sheep, and pigs); F and G have been 60
reported from felines and rats respectively (Monis et al., 2003). More recently, Assemblage H has 61
been detected in marine vertebrates (Lasek-Nesselquist et al., 2010). Recent studies throughout the 62
world have demonstrated that calves in dairy and beef herds may harbour more than one genotypes 63
of G. duodenalis (Trout et al., 2004, 2005; Itagaki et al., 2005; Lalle et al., 2005; Mendonca et al., 64
2007; Langkjaer et al., 2007; Winkworth et al., 2008). However, although the World Health 65
4 Organization has considered G. duodenalis to have a zoonotic potential for around 30 years (WHO, 66
1979), direct evidence has been lacking.
67
A significant prevalence of Giardia isolates has been earlier reported in Indian dairy cattle 68
based on microscopic findings (Deshpande and Shastri, 1981). However, surprisingly, there have 69
been no prior molecular characterization studies of Giardia in Indian cattle although extensive 70
molecular epidemiological studies have been carried out in a number of countries. There is also 71
lack of data required for the assessment of zoonotic transmission of G. duodenalis between cattle 72
and humans in India. Consequently, this study has been formulated to understand the public health 73
significance of this parasite from cattle and get a clearer epidemiological picture, with better 74
information on the zoonotic potential and transmission mechanisms for humans.
75 76 77
5 2. Materials and Methods
78
2.1. Collection of fecal samples 79
Bovine fecal samples used in this study were collected from 180 dairy cattle including 40 80
pre-weaned calves (0-2 months old), 72 post-weaned calves (3-12 months old) and 68 heifers and 81
adults (> 12 months) with and without diarrhoea from two dairy farms: the Harringhata Cattle 82
Farm, Nadia and Ramakrishna Mission Dairy Farm, Narendrapur, 24 Parganas (N), West Bengal, 83
India from October 2008 to August 2009. Feces were collected directly from the rectum of each 84
animal with a gloved hand and transferred into sterile wide mouthed, labeled plastic containers and 85
immediately placed into an insulated container packed with ice or cold packs. In addition to these, 86
stool samples were also collected from 51 dairy farm workers of these two farms who were in 87
direct or indirect contact with these animals but showed no visible clinical signs of giardiasis.
88
Specimens were transported to the Division of Parasitology, National Institute of Cholera and 89
Enteric Diseases, Kolkata, India as early as possible and processed within two days of collection.
90
Three aliquots of each sample were frozen without preservative in 1.5 ml cryovials at -80 °C for 91
ELISA and PCR studies.
92 93
2.2. Parasite Detection 94
Microscopic examination was performed on all samples within 48 hours after collection.
95
Three separate techniques were used for the detection of G. duodenalis in the fecal samples. Firstly 96
iodine wet mount staining and secondly Trichrome stain were performed for the detection of 97
trophozoites and cysts of Giardia according to the Centers for Disease Control and Prevention 98
(CDC) method (http://www.dpd.cdc.gov/dpdx/HTML/DiagnosticProcedures.htm). For microscopic 99
screening, parasite cysts and oocysts present in fecal samples were concentrated using a FPC®
100
Fecal Parasite Concentrator (Evergreen Scientific, Los Angeles, CA, USA).
101
6 Antigen capture Enzyme Linked Immunosorbent Assay (ELISA) was also performed with 102
all the frozen samples for detection of Giardia using a commercially available kit GIARDIA II 103
(TECHLAB, Blacksburg, VA, USA). The monoclonal antibody based ELISA test was used as 104
instructed by the manufacturer.
105 106
2.3. DNA extraction 107
Genomic DNA was extracted from frozen samples from individuals that were positive by 108
microscopy and ELISA using the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, CA, USA) 109
according to the manufacturer’s instructions except that the stool lysis temperature was increased to 110
80 °C. The eluted DNA was quantified spectrophotometrically and stored at -20 °C for further PCR 111
studies.
112 113
2.4. PCR Analysis 114
For the detection of Giardia, amplification of a fragment of the β-giardin gene was 115
performed using primers described by Caccio et al. (2002) and Lalle et al. (2005). A 753 bp 116
fragment was amplified in the primary PCR reaction using the forward primer G7 and reverse 117
primer G759. In the secondary PCR reaction a 511 bp fragment was amplified using the forward 118
primer Glbg511F (5´-GAACGAACGAGATCGAGGTCCG-3´) and the reverse primer Glbg511R 119
(5´-CTCGACGAGCTTCGTGTT-3´). PCR mixtures and cycling conditions were identical to those 120
previously described (Lalle et al., 2005) except that the primary PCR reaction mixture also 121
contained non-acetylated BSA (New England Biolabs, Beverly, MA, USA) to a final concentration 122
of 0.1 µg/µl. All PCR products were analysed by 1.5% agarose gel electrophoresis and visualised 123
after ethidium bromide staining.
124 125
2.5. Restriction fragment length polymorphism (RFLP) analysis 126
7 Secondary PCR products were purified using the High Pure PCR product purification kit 127
(Roche Diagnostics, Mannheim, Germany). The restriction analysis was performed as described 128
before (Lalle et al., 2005). Briefly, 10 µl of the nested PCR products of the β-giardin gene were 129
digested with 10 units of restriction endonuclease HaeIII (New England Biolabs) and 2 µl of 10×
130
buffer in a total volume of 20 µl, at 37 °C for 3 hours. Restriction products were fractionated on a 131
2% agarose gel and visualised after ethidium bromide staining.
132 133
2.6. DNA Sequencing and Phylogenetic Analysis 134
In order to determine G. duodenalis subgenotypes and to confirm the PCR-RFLP results, all 135
purified secondary PCR products that were positive for Giardia were directly sequenced in both 136
directions using an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA, 137
USA) with forward and reverse primers. Sequences obtained were analyzed and assembled using 138
CLUSTAL W software (Higgins et al., 1994). The obtained nucleotide sequences were used to 139
search the GenBank nucleotide sequence database for sequence similarities using BLAST software 140
(NCBI, Bethesda, MD, USA). Multiple alignments of these sequences were made using the BioEdit 141
program (Hall, 1999).
142
For comparative phylogenetic analysis, reference sequences retrieved from the GenBank 143
were aligned with the representative sequences of each species or genotype of Giardia obtained in 144
this study and a neighbor-joining tree was constructed using TREECON for Windows version 1.3b 145
(Van de Peer and De Wachter, 1994). Distance estimations were carried out using the Jukes and 146
Cantor correction. The branch reliability of the neighbor-joining tree was assessed by the bootstrap 147
method with 1000 replications. The nucleotide sequence of Giardia muris (GenBank accession no.
148
EF455599) was used as an outgroup to root the neighbor-joining tree since the construction of an 149
unrooted tree showed it to be the most divergent member under analysis.
150 151 152
8 3. Results
153
The overall prevalence of Giardia in cattle was 12.2% (22/180, Table 1); 27.5% (11/40) 154
pre-weaned calves, 12.5% (9/72) post-weaned calves and 2.9% (2/68) heifers and adults were 155
positive for Giardia by both microscopy and ELISA. Among dairy farm workers, Giardia was 156
present in 27.4% (14/51) of the screened samples. Successful PCR amplification of the Giardia β- 157
giardin gene was accomplished for all the samples positive by microscopy and ELISA.
158 159
3.1. Genotyping and Phylogenetic Analysis of Giardia 160
Among cattle, PCR-RFLP results identified G. duodenalis Assemblage E as the most 161
prevalent genotype although a substantial number of the isolates from dairy calves were found to be 162
Assemblage A (Table 1). Interestingly, Giardia isolates from one pre-weaned and two post-weaned 163
calves produced four visible bands (201, 186, 150 and 110 bp) upon restriction digestion of the β- 164
giardin nested PCR products by HaeIII (Fig. 1). This meant that there was a mixed G. duodenalis 165
Assemblage A and E infection in these calves since RFLP analysis of Assemblage A would have 166
produced three visible bands (201, 150 and 110 bp) while Assemblage E would have generated 167
three bands visible at 186, 150 and 110 bp, upon restriction analysis by HaeIII (Lalle et al., 2005).
168
DNA sequencing of most Assemblage E positive PCR products (10/14) showed a mismatch of two 169
base pairs with the reference sequences for subtypes E1 (GenBank accession no. AY072729), E2 170
(GenBank accession no. AY545650) and E3 (GenBank accession no. AY653159), the mismatches 171
being at two different positions for each subtype. However, E1 and E2 were also detected in a few 172
samples (Table 2). Additionally, all the Assemblage A positive isolates in cattle were identified as 173
subtype A1 by DNA sequencing of nested PCR products of the β-giardin gene.
174
G. duodenalis Assemblage B (57.1%, 8/14) was more prevalent than Assemblage A 175
(42.9%, 6/14) in dairy farm workers (Table 1). DNA sequencing of the nested PCR products 176
9 identified A1 and B3 as the dominant subtypes although a number of other subtypes were also 177
found in a few samples (Table 2).
178
Under phylogenetic analysis, all the G. duodenalis assemblages studied (A-F) formed an 179
individual distinct cluster (Fig. 2). Grossly, two major groups were formed. One group comprised 180
of assemblages A, E and F while the other one contained assemblages B, C and D. This indicated 181
that assemblages A, E and F were related to each other and that assemblages B, C and D were 182
related to each other. Intra-Assemblage genetic polymorphism was also evident within assemblages 183
A, B and E.
184 185
3.2. Nucleotide sequence accession numbers 186
Representatives for genotypes of G. duodenalis identified in this study have been submitted 187
to GenBank under the accession numbers: GQ290390, GQ345009 and GQ345010.
188 189 190
10 4. Discussion
191
There has been considerable interest in recent years in the potential for zoonotic 192
transmission of G. duodenalis particularly with respect to cattle and other livestock. Still, until now 193
not even a single study has examined the genetic diversity of G. duodenalis in Indian dairy cattle.
194
Also, there is lack of information regarding the zoonotic potential of this parasite for human beings 195
working at dairy farms in a developing country like India. The environment at dairy farms in India 196
is such that close contact of humans with animals occurs regularly, putting dairy farm workers, 197
cattle handlers and veterinarians at risk of contracting zoonotic diseases. This study helps to 198
provide valuable information on both aspects viz. the genetic diversity and the zoonotic potential of 199
G. duodenalis from Indian dairy cattle.
200
The present study represents the first report on the molecular characterization of G.
201
duodenalis in Indian cattle. Overall, Giardia was detected in 12.2% (22/180) of the bovine fecal 202
samples screened by microscopy, ELISA and PCR studies. The relatively higher prevalence of 203
Giardia infection found in a previous study in Indian cattle (Deshpande and Shastri, 1981) can be 204
explained by the fact that only calves were sampled in that study whereas both calves and adult 205
cattle were screened for Giardia in the current study. Further, results from the present study 206
indicate that infections by G. duodenalis are more prevalent in calves than in adult cattle, which 207
agrees with previous reports (Huetink et al., 2001; Olson et al., 2004; Mendonca et al., 2007). A 208
significant number of Assemblage E isolates detected in calves and adult cattle were shown to be 209
genetically and phylogenically different from all the previous known Assemblage E subtypes 210
namely E1, E2 and E3 (Lalle et al., 2005); however, they were relatively more closely related to E1 211
and E3 under phylogenetic analysis. This refers a novel subgenotype of G. duodenalis Assemblage 212
E in Indian cattle. As expected, this livestock-specific assemblage was found more frequently in 213
cattle sampled in this study, although the most common zoonotic genotype Assemblage A 214
(Thompson et al., 2000) was also identified in a number of calves. Similar observations have been 215
11 reported in previous studies from across the globe (O’Handley et al., 2000; Huetink et al., 2001;
216
Trout et al., 2004, 2005; Itagaki et al., 2005; Mendonca et al., 2007; Langkjaer et al., 2007).
217
Additionally, it was also observed that prevalence of Assemblage A was more or less equal in pre- 218
weaned as well as post-weaned calves. Thus no age-related distribution of zoonotic genotypes of 219
Giardia was found in this study which is similar to findings from previous studies (Trout et al., 220
2004, 2005; Langkjaer et al., 2007).
221
Results of this study provide useful molecular evidence on the zoonotic transmission of G.
222
duodenalis between cattle and dairy farm workers. DNA sequencing of nested PCR products of the 223
β-giardin gene genotyped Assemblage A isolates from all calves and most dairy farm workers as 224
subtype A1. It is interesting to note here that the two most common subtypes of Assemblage A, A1 225
and A2, differ significantly in host preference; animals are mostly infected with A1 whereas 226
humans are mostly infected with A2 (Xiao and Fayer, 2008). Therefore, a relatively higher 227
prevalence of A1 in comparison to A2 found in humans in this study is of zoonotic importance. In 228
addition, mixed G. duodenalis Assemblage A and E infections were also detected in three calves in 229
the present study which suggests that calves, although primarily infected with G. duodenalis 230
Assemblage E, are frequently hosts of the zoonotic G. duodenalis Assemblage A. This is of 231
potential public health significance since calves infected with Giardia normally shed a large 232
number of cysts in their faeces (O’Handley et al., 1999) and could thus act as a potential zoonotic 233
reservoir for human giardiasis especially on the dairy farm premises. However, Assemblage B was 234
not detected in cattle in the present study although it was the most prevalent genotype found in 235
dairy farm workers thus indicating anthroponotic transmission of this genotype is more common.
236
In conclusion, results of this study provide new insights into the epidemiology and genetic 237
diversity of Giardia duodenalis in Indian dairy cattle. Additionally, results also point towards a 238
possible zoonotic transmission of this parasite between cattle and human workers on dairy farms in 239
India. Even a few calves infected with zoonotic genotypes of Giardia could pose a significant 240
12 public health risk directly to handlers or indirectly as an important reservoir for human waterborne 241
outbreaks of giardiasis.
242 243 244
13 Acknowledgements
245
This study was supported partially by grants from (i) Okayama University Program of Founding 246
Research Centre for Emerging and Reemerging Infectious Disease, Ministry of Education, Culture, 247
Sports, Science and Technology of Japan, (ii) The Japan Health Sciences Foundation 248
and (iii) US Embassy in India and Emerging and Re-emerging Infectious Disease and Disease 249
Surveillance (ERIDDS), USA and Centers for Disease Control and Prevention, Atlanta, USA. The 250
authors acknowledge Dr. Altaf Lal, Health Attaché and HHS Regional Representative for South 251
Asia, U.S. Embassy New Delhi for his constructive suggestions, comments, support and immense 252
help throughout the entire study; Prof. Y. Takeda and Dr. G. B. Nair for their continuous 253
constructive suggestions, support and critical review during this study; and Debarati Ganguly of 254
Calcutta University for her careful proof reading and correction of English in the manuscript.
255
Authors also acknowledge Mr. Avik Kumar Mukherjee and Mr. Arjun Ghosh of Ganguly lab for 256
their technical discussion during the study.
257
None of the authors have any financial interest in any commercial company represented in this 258
study, nor any other potential conflicts of interest.
259 260 261
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17 Figure Captions
335
Figure 1. Genotyping of Giardia isolates by RFLP analysis based on digestion of β-giardin gene 336
PCR products by HaeIII. Lanes 1 and 5: 100 bp plus DNA ladders, lane 2: Assemblage E, lane 3:
337
Mixed Assemblage A and E, lane 4: Assemblage A (bovine source), lane 6: Assemblage A (human 338
source) and lane 7: Assemblage B.
339 340
Figure 2. Phylogenetic relationship among Giardia isolates as inferred by neighbor-joining analysis 341
of the partial β-giardin nucleotide sequences. Bootstrap values above 50% out of 1000 replicates 342
are indicated at each node. Accession numbers for sequences obtained from GenBank are given in 343
parentheses. The sequence of Giardia muris was used as an outgroup.
344 345
18 Tables
346
Table 1. Detection of different genotypes of Giardia duodenalis by PCR-RFLP of the β-giardin 347
gene in fecal samples collected from different age groups of dairy cattle and dairy farm workers.
348 349
Source Sample size
Giardia duodenalis
A B E A + E
Pre-weaned calves
40 3 0 7 1
Post-weaned calves
72 2 0 5 2
Heifers and Adult Cattle
68 0 0 2 0
Dairy Farm Workers
51 6 8 0 0
350 351
19 Table 2. Giardia duodenalis assemblages A, B and E subtypes identified in dairy cattle and dairy 352
farm workers by DNA sequencing of the β-giardin gene.
353 354
Host Cattle Human
Assemblage A E A B
Subtype A1=5a
A+E=3b
En=10c
E1=3
E2=1
A1=4
A2=2
B1=1
B3=6
B4=1
a Number refers to the isolates detected 355
b Subtypes could not be determined 356
c En refers to the novel subtype detected 357
358
Figure
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Figure
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